1. Field of the Disclosure
This invention relates generally to proteins and their production in recombinant host cells. More particularly, it relates to polynucleotide sequences that encode novel paired basic amino acid converting enzymes (generically referred to as "PACE," with the two new enzymes being PACE 4 (SEQ ID NO:1) and 4.1 (SEQ ID NO:3)), to the production of PACE 4 and 4.1 in transformed cells, and to compositions comprising the PACE 4 and 4.1 enzymes. Materials and methods are also provided for the production of mature forms of proteins from heterologous precursor polypeptides using PACE 4 or 4.1, which are expressed in recombinant host cells.
2. Description of Related Art
Many eukaryotic proteins synthesized in bacteria fold incorrectly and, consequently, exhibit low specific activities. Post-translational proteolysis is a common mechanism required for the synthesis of biologically active proteins and peptides in all eukaryotes examined, including yeast (R. S. Fuller et al., (1988), Ann. Rev. Physiol. 50:345), invertebrates (R. H, Scheller et al. (1983), Cell 32:7), and mammalian cells (J. Douglass et al. (1984), Ann. Rev. Biochem. 53:665; W. S. Sossin et al. (1989), Neuron 2, 1407).
One of the early events in precursor protein maturation is endoproteolysis at the carboxyl side of pairs of basic amino acid sequences (e.g. -LysArg- and -ArgArg-). This kind of endoproteolytic cleavage was initially inferred from the sequences of several endocrine and neuroendocrine precursor proteins and was first proposed from studies of proinsulin (D. F. Steiner et al. (1968), Science 157:697; R. E. Chance et al. (1968), Science 161:165) and the ACTH/.beta.-endorphin precursor, proopiomelanocortin (POMC)(M. Chretien and C. H. Li (1967), Can. J. Biochem. 45:1163). Subsequent studies have revealed a broad spectrum of precursor proteins that require endoproteolysis at pairs of basic amino acids to yield mature peptides including serum factors (A. K. Bentley et al. (1986), Cell 45:343), viral proteins (C. M. Rice et al. (1986), Virology 151:1; C. M. Rice et al. (1985), Science 229:726; J. M. McCune et al. (1988), Cell 53:55), growth factors (L. E. Gentry et al. (1988), Mol. Cell Biol. 8:4162; K. Sharples et al. (1987), DNA 6:239; M. Yanagisawa et al. (1988), Nature 332:411; and Gray et al. (1983), Nature 303:722) and receptors (Y. Yosimasa (1988), Science 240:784).
Several candidate enzymes that are capable of cleaving at single or paired basic residues in vitro have been proposed as authentic mammalian precursor endoproteases. See, for example, Y. P. Loh and H. Gainer, in Brain Peptides, D. T. Krieger, M. J. Brownstein, J. B. Martin, Eds. (Wiley-Interscience, New York, 1983), pp.76-116; M. Chretien, et al. in Cell Biology of the Secretory Process (Karger, Basel, Switzerland, 1983), pp.214-246; A. J. Mason, et al. (1983), Nature 303:300; P. J. Isackson et al. (1987), J. Cell. Biochem. 33:65; I. Lindberg et al., (1984), J. Neurochem 42:1411; J. A. Cromlish et al. (1986), J. Biol. Chem. 261:10850; K. Docherty et al. (1984), J. Biol. Chem. 259:6041; T. C. Chang and Y. P. Loh (1984), Endocrinology 114, 2092; B. P. Noe et al. (1984), J. Cell. Biol. 99:578; U. P. Loh (1986), J. Biol. Chem. 261:11949; H. W. Davidson et al. (1987), Biochem. J. 246:279; P. Gluschankof et al. (1987), J. Biol. Chem. 262:9615; C. Clamigrand et al. (1987), Biochem 26:6018; S. O. Brennan and R. J. Peach (1988), FEBS Letters 229:167; R. S. Fuller et al. (1989), Proc. Natl. Acad. Sci. USA 86:1434; K. Mizuno et al. (1989), Biochem. Biophys. Res. Comm. 159:305; I. C. Bathurst et al. (1987), Science 235:348; and G. Thomas et al. (1988), Science 241:226. However, none of these candidate activities have been shown to be a bona fide precursor cleaving endoprotease in vivo.
The yeast enzyme Kex2, a membrane-bound, Ca.sup.2+ -dependent serine protease (K. Mizuno et al. (1988), Biochem. Biophys. Res. Commun. 156:246; R. S. Fuller et al. (1989), Proc. Natl. Acad. Sci. USA 86:1434), has been considered to be a prototypic proprotein convertase. The Kex2 endoprotease, which is encoded by the KEX2 gene, functions late in the secretory pathway of Saccharomyces cerevisiae, and cleaves the polypeptide chains of prepro-killer toxin and prepro-.alpha.-factor at the paired basic amino acid sequences Lys-Arg and Arg-Arg (D. Julius et al. (1984), Cell 36:309). Furthermore, co-expression of the KEX2 gene with POMC in BSC-40 cells (a cell line which is incapable of processing this peptide precursor) resulted in the generation, by proteolysis at pairs of basic amino acids, of authentic product peptides, including .beta.-LPH and .beta.-endorphin (Thomas et al. (1988), id.).
Two human DNA sequences, fur and PC2, share some structural homology with each other and with the KEX2 gene sequence. A. M. W. van den Ouweland et al. (1990), Nucleic Acids Res. 18:664 (presenting a cDNA coding sequence for fur; the cDNA sequence of FIG. 1 differs from this sequence in the region encoded by nucleotides 1-238); R. S. Fuller et al., Science 246:482 (1989); S. P. Smeekens (1990), J. Biol. Chem. 265:2997. The fur locus was initially identified by its proximity to the fes/fps proto-oncogene (A. J. M. Roebroek et al. (1986), EMBO J. 5:2197). PC2 was identified by amplification of a human insulinoma library by the polymerase chain reaction using KEX2-derived primers; it shares a partial homology with Kex2, especially in the putative active site domains (S. P. Smeekens and D. F. Steiner, (1990) J. Biol. Chem. 265:2997).
Another related cDNA encoding a protein variously called PC1 (Seidah et al. (1991) Mol. Endocrinol. 5:111-122) or PC3 (Smeekens et al. (1991) Proc. Natl. Acad. Sci. USA 88:340-344) has been isolated from murine pituitary cells. PC1/PC3 also shares amino acid similarity with KEX2, particularly in the putative catalytic domains.
Recently a functional activity has been demonstrated for PC2 and PC1/PC3, using as a substrate the prohormone proopiomelanocortin (POMC). In the anterior lobe of the pituitary, POMC is processed to yield the hormones ACTH, .beta.-lipotropin, and .beta.-endorphin, while in the intermediate lobe it is processed to yield .alpha.-melanocyte stimulating hormone (.alpha.-MSH) and variant forms of ACTH and .beta.-lipotropin. PC1/PC3 expressed in processing deficient cells cleaved POMC to give ACTH, but was less efficient in cleaving .beta.-lipotropin to give .beta.-endorphin. This is similar to the activity found in pituitary corticotrophic cells. Coexpression of PC2 and PC1/PC3 efficiently converted .beta.-lipotropin, as is found in melanotrophic cells. Coexpression of POMC and PC2 in adrenomedullary chromaffin cells resulted in secretion of .beta.-endorphin and .alpha.-MSH. Therefore PC1/PC3 and PC2 have differing substrate selectivities which yield physiologically relevant processed polypeptides.